DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin’s Inhibitory Effect

Abstract Amino acid substitutions at nonconserved protein positions can have noncanonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

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Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

10.26686/wgtn.17147804.v1 ◽

2021 ◽

Author(s):

◽

Janine Sharma

Keyword(s):

Dna Binding ◽

Molecular Biology ◽

Active Form ◽

Binding Domain ◽

Dna Ligase ◽

Dna Binding Domain ◽

Chloramphenicol Resistance ◽

T4 Dna Ligase ◽

Dna Ligases ◽

Lead Candidate

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

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Long-Range Repression by Multiple Polycomb Group (PcG) Proteins Targeted by Fusion to a Defined DNA-Binding Domain in Drosophila

Genetics ◽

10.1093/genetics/158.1.291 ◽

2001 ◽

Vol 158 (1) ◽

pp. 291-307

Author(s):

Robin R Roseman ◽

Kelly Morgan ◽

Daniel R Mallin ◽

Rachel Roberson ◽

Timothy J Parnell ◽

...

Keyword(s):

Dna Binding ◽

Long Range ◽

Binding Domain ◽

Polycomb Group ◽

Dna Binding Domain ◽

Interaction Domain ◽

Bona Fide ◽

Biochemical Analyses ◽

Chromosomal Loci

Abstract A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

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Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

10.26686/wgtn.17147804 ◽

2021 ◽

Author(s):

◽

Janine Sharma

Keyword(s):

Dna Binding ◽

Molecular Biology ◽

Active Form ◽

Binding Domain ◽

Dna Ligase ◽

Dna Binding Domain ◽

Chloramphenicol Resistance ◽

T4 Dna Ligase ◽

Dna Ligases ◽

Lead Candidate

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

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1HN, 13C, and 15N backbone resonance assignments of the human DNA ligase 3 DNA-binding domain (residues 257-477)

Biomolecular NMR Assignments ◽

10.1007/s12104-019-09896-9 ◽

2019 ◽

Vol 13 (2) ◽

pp. 305-308

Author(s):

Braden M. Roth ◽

Kristen M. Varney ◽

Hui Yang ◽

David J. Weber ◽

Alan E. Tomkinson

Keyword(s):

Dna Binding ◽

Resonance Assignments ◽

Binding Domain ◽

Dna Ligase ◽

Dna Binding Domain ◽

Backbone Resonance Assignments ◽

Backbone Resonance ◽

Human Dna

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Substitutions at Non-Conserved Rheostat Positions Modulate Function by Re-Wiring Long-Range, Dynamic Interactions

10.1101/2020.07.02.185207 ◽

2020 ◽

Cited By ~ 1

Author(s):

Paul Campitelli ◽

Liskin Swint-Kruse ◽

S. Banu Ozkan

Keyword(s):

Dna Binding ◽

Long Range ◽

Protein Function ◽

Binding Domain ◽

Dna Binding Domain ◽

Dynamic Coupling ◽

Long Distance ◽

Modulate Function ◽

Functional Changes ◽

Wide Range

AbstractAmino acid substitutions at nonconserved protein positions can have non-canonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities (“DFI”) and dynamic coupling (“DCI”) for positions in the linker region of the lactose repressor protein (“LacI”). This region contains nonconserved positions for which substitutions alter DNA binding affinity. We first chose to study eleven substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA binding positions, and the degree of flexibility change correlated with experimentally-measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA binding positions in a manner that captured other experimentally-determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated almost no substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type LacI, the dynamic couplings between the DNA binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and non-canonical substitutions outcomes at nonconserved positions arise from re-wiring long-range communication among functionally-important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

Download Full-text